Natural glucocorticoids are steroid hormones that regulate a variety of biological processes and influence many physiological functions by virtue of their diverse roles in the growth, development, differentiation, and maintenance of basal and stress-related homeostasis (Munck, 1984; Clark, 1992). Glucocorticoids affect probably every organ in the mammalian body, yet many of their effects are specific for certain cell types or tissues. In addition, synthetic glucocorticoids are among the most widely prescribed drugs worldwide, used primarily as anti-inflammatory and immunosuppressive agents.
Steroid resistance or steroid dependence is still a major clinical concern for a large number of patients afflicted with inflammatory diseases as current therapies rely on the use of potent immunomodulators that can induce serious side-effects. Abnormalities in glucocorticoid sensitivity can be divided into 2 major groups: resistance and hypersensitivity. Resistance to glucocorticoids is characterized by the inability of organism or target tissues to respond to steroid molecules and can be generalized or tissue-specific, transient or permanent, partial or complete, and compensated or non-compensated (Chrousos, 1982; Chrousos, 1993). Complete glucocorticoid resistance is not compatible with life, given that absence of functional glucocorticoid receptors (GRs) in GR−/−knockout mice leads to severe neonatal respiratory distress syndrome and death within a few hours after birth (Cole, 1995).
Treatment with glucocorticoids is the most potent therapy available for acute and chronic asthma especially for patients with severe disease. Unfortunately, a certain fraction of asthmatics are steroid resistant (SR) and do not benefit from standard treatment. A rough estimate is that SR asthma occurs in approximately 15-20% of the asthmatic population. It is critical to identify these patients as early as possible. Patients who do not respond to low steroid doses are often placed on higher doses, which in SR asthmatics can cause significant adverse effects without providing significant benefit (Leung, 1995).
Inflammatory disorders normally treated with the natural or synthetic glucocorticoids, comprise asthma, rheumatoid arthritis, ulcerative colitis, Crohn's disease and other disorders. An inflammatory disorder includes complex diseases, which involve many factors and cell types and have a distinct inflammatory cytokine profile. The nature and magnitude of an immune response is largely dictated by the profile of the foreign antigen to which the immune system has been exposed. This event sets into motion a series of events that ultimately lead to the generation of humoral and cell-mediated immunity. These two different effector functions are brought about by the presence of two subpopulations of helper T cells (Th1 and Th2). Under “normal” healthy conditions, there is a delicate balance between the cytokines produced by the cell types Th1 and Th2. If this balance is lost, there will be a polarization resulting in predominantly Th1 or Th2 type inflammation and clinical manifestation of the disease will occur. As also indicated, different inflammatory diseases can be segregated as being either Th1 or Th2, depending on the cytokine profile seen. The cytokine picture indicates that asthma and ulcerative colitis are Th2 type diseases, while rheumatoid arthritis is associated with a Th1 type of inflammation.
New therapeutics have been applied to restore the “in-balance” in Th1 type diseases by reducing the cytokine profile of Th1 and thereby allowing an increase of the Th2 profile (Neurath et al., 1995; Mannon et al., 2004). The bacterial DNA has been shown to have immune stimulatory effects capable of activating B cells and natural killer cells (reviewed in Krieg, 1998) due to the presence of unmethylated CpG dinucleotides (CpG motifs). The vertebrate immune system has evolved the ability to recognize unmethylated CpG motifs and respond with a rapid and coordinated cytokine response leading to the induction of humoral and cell-mediated immunity (Krieg, 1996). For example, human and mouse cells respond to oligonucleotides containing a CpG motif by enhanced secretion of interferon-gamma (IFN-gamma) (Iho et al., 1999: Cowdery et al., 1996), IL-1, IL-6, IL-12 and tumor necrosis factor alpha (TNF-alpha) (Stacey et al., 1996; Jakob et al., 1998; Sparwasser et al., 1998). Due to the nature of cytokines induced, CpG containing oligonucleotides are largely considered to induce a Th1 profile both in vitro and in vivo (Zimmermann et al., 1998; Kline, 2000). In addition to the presence of CpG motifs, researchers have also noted that synthesizing CpG oligonucleotides with a full nuclease-resistant phosphorothioate backbone can potentate the stimulatory effects of the oligonucleotides, probably via stimulation of B-cells, whereas the same sequence with native phosphodiester backbone had no effect (Zhao et al., 1996).
The use of CpG motifs containing oligodeoxynucleotides (ODNs), or DNA vaccination which induce allergen-specific or unspecific Th1 responses are currently considered as a strategy both for the prevention and therapy of asthma (Wolleben, 2006).
The international patent application WO 2007/004977 concerns the treatment of a steroid refractory or steroid dependent patient afflicted with an inflammatory condition not responding or responding poorly or inadequately to a given anti-inflammatory treatment. The steroid efficacy can be enhanced by administering an effective amount of an oligonucleotide having the sequence 5′-Xm-CG-Yn-3′ to the patient. In the sequence of the oligonucleotide X is A, T, C or G, Y is A, T, C or G, m=1- 100, n=1-100 and at least one CG dinucleotide is unmethylated.
The international patent application WO 2007/004979 concerns a method for enhancing the steroid efficacy in a steroid refractory or steroid dependent patient afflicted with an inflammatory condition not responding or responding poorly or inadequately to a given anti-inflammatory treatment by administering an effective amount of an oligonucleotide having the sequence 5′-TTCGT-Yn-3′ to the patient. In the oligonucleotide sequence, X is A, T, C or G, Y is A, T, C or G, m=1-7, n=1-7 and at least one CG dinucleotide is unmethylated.
The above methods are applicable also in a situation, where weaning down the dosing of the anti-flammatory treatment is ineffective. Another therapy involving the use of oligonucleotides is presented in the international application WO 2002/085308, which discloses compositions, formulations, and kits for the treatment of respiratory and pulmonary diseases including asthma, infectious diseases, cancer and diseases having secondary effects on the lungs. This indicates that the compositions containing both anti-sense oligonucleotides and steroid agents and/or ubiquinones have effects superior to each agent alone and may be used as preventative, prophylactic or therapeutic single therapies or in conjunction with other therapies. The anti-sense oligonucleotide preferably contains about 0-15% of adenosine (A) and is anti-sense to the initiation codon, the coding region, the 5′-end or the 3′-end genomic flanking regions, the 5′ or 3′ intron-exon junctions, or regions within 2-10 nucleotides of the junctions of at least one gene regulating or encoding a target polypeptide associated with lung or airway dysfunction or cancer, or that is anti-sense to the corresponding mRNA.
The multi-gene approach disclosed in the international patent application WO 2004/001073, provides specific marker genes, which allow discrimination of inflammatory bowel disease, ulcerative colitis and Crohn's disease. The method compares gene expression profiles in biopsy samples obtained from inflamed, and optionally also from non-inflamed, areas in the intestines.
U.S. 20040197786 presents a method for examining steroid responsiveness in atopic dermatitis patients. In the method the expression levels of the genes RING6 and HLA-DMB are suggested as markers for testing steroid responsiveness and for use in the screening for compounds that may be used to improve steroid responsiveness.
The international patent application WO2003/021261 concerns a method for predicting the efficacy of a drug for treating an inflammatory disease, by analyzing the gene expression profile in a sample isolated from the patient.
Steroid dependency and steroid resistance does also occur in other conditions, such as steroid dependent nephrotic syndrome (SDNS), steroid-dependent corneal inflammation, edema of various etiology etc.
Despite the multitude of available therapies, the individual variations to said therapies remains a challenge to a physician confronting a patient presenting with syndromes related to a disease with no response or a poor response to a given steroid therapy. It would be advantageous for a physician dealing with said problems to have a simple and rapid in vitro test, which would enable a relatively reliable prediction of the response of the individual patient to the chosen therapy.
Therefore, there is a great demand for a simple, straightforward and rapid in vitro method for predicting steroid efficacy in a steroid unresponsive individual and to determine if the steroid efficacy in a steroid unresponsive, i.e. steroid resistant or steroid dependent individual can be enhanced. A method for predicting the response would simplify the choice of anti-inflammatory treatment, help to ameliorate the disease in question and decrease the costs and detrimental side effects of the steroid therapy, thereby increasing the quality and length of life for a large number of patients.